The long-term goal of this proposal is to elucidate the molecular mechanism(s) by which the Wingless (Wg) morphogen gradient and signaling are regulated during development. Drosophila Wg encodes an evolutionarily conserved glycoprotein of the Wnt family. Aberant Wg/Wnt signaling activity underlies a number of human developmental disorders and contributes to a variety of cancers. Thus, elucidation of the Wg/Wnt signaling pathway will provide new insights into the mechanisms of related human diseases. Although the components of the Wg/Wnt signaling pathway in its receiving cells have been well characterized over the past 20 years, it remains poorly understood how the mature form of Wg is secreted and travels to its receiving cells to form a concentration gradient, and how the Wg signaling activates the transcription of its downstram target genes. This is an application for continuing support. The current proposal will examine the mechanisms of Wg secretion and its signaling activity in the regulation of the transcription of its target genes. Fist, we will determine the functions of three newly identified ER and Golgi vesicle proteins in Wg secretion (Aim1). Second, we will examine the role(s) of ubiquitin E3 ligase Su (dx) and the deubiquitinating enzyme USP8 in Wls ubiquitination and Wg secretion (Aim2). Third, we will examine the function of Drosophila UBR5 in regulating Wg signaling (Aim3). Taken together, our genetic and biochemical analyses of the newly identified genes in the Wg pathway will provide new insights into the mechanisms of Wg secretion and the transcriptional activation of Wg target genes. Since the Wg signaling transduction pathway is conserved between Drosophila and humans, the outcomes of this project will clearly provide new insights into the mechanisms of various disease processes including cancers.